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  1. Gallagher MT, Cupples G, Ooi EH, Kirkman-Brown JC, Smith DJ
    Hum Reprod, 2019 07 08;34(7):1173-1185.
    PMID: 31170729 DOI: 10.1093/humrep/dez056
    STUDY QUESTION: Can flagellar analyses be scaled up to provide automated tracking of motile sperm, and does knowledge of the flagellar waveform provide new insight not provided by routine head tracking?

    SUMMARY ANSWER: High-throughput flagellar waveform tracking and analysis enable measurement of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses, which are not possible by tracking the sperm head alone.

    WHAT IS KNOWN ALREADY: The clinical gold standard for sperm motility analysis comprises a manual analysis by a trained professional, with existing automated sperm diagnostics [computer-aided sperm analysis (CASA)] relying on tracking the sperm head and extrapolating measures. It is not currently possible with either of these approaches to track the sperm flagellar waveform for large numbers of cells in order to unlock the potential wealth of information enclosed within.

    STUDY DESIGN, SIZE, DURATION: The software tool in this manuscript has been developed to enable high-throughput, repeatable, accurate and verifiable analysis of the sperm flagellar beat.

    PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the software tool [Flagellar Analysis and Sperm Tracking (FAST)] described in this manuscript, we have analysed 176 experimental microscopy videos and have tracked the head and flagellum of 205 progressive cells in diluted semen (DSM), 119 progressive cells in a high-viscosity medium (HVM) and 42 stuck cells in a low-viscosity medium. Unscreened donors were recruited at Birmingham Women's and Children's NHS Foundation Trust after giving informed consent.

    MAIN RESULTS AND THE ROLE OF CHANCE: We describe fully automated tracking and analysis of flagellar movement for large cell numbers. The analysis is demonstrated on freely motile cells in low- and high-viscosity fluids and validated on published data of tethered cells undergoing pharmacological hyperactivation. Direct analysis of the flagellar beat reveals that the CASA measure 'beat cross frequency' does not measure beat frequency; attempting to fit a straight line between the two measures gives ${\mathrm{R}}^2$ values of 0.042 and 0.00054 for cells in DSM and HVM, respectively. A new measurement, track centroid speed, is validated as an accurate differentiator of progressive motility. Coupled with fluid mechanics codes, waveform data enable extraction of experimentally intractable quantities such as energy dissipation, disturbance of the surrounding medium and viscous stresses. We provide a powerful and accessible research tool, enabling connection of the mechanical activity of the sperm to its motility and effect on its environment.

    LARGE SCALE DATA: The FAST software package and all documentation can be downloaded from www.flagellarCapture.com.

    LIMITATIONS, REASONS FOR CAUTION: The FAST software package has only been tested for use with negative phase contrast microscopy. Other imaging modalities, with bright cells on a dark background, have not been tested but may work. FAST is not designed to analyse raw semen; it is specifically for precise analysis of flagellar kinematics, as that is the promising area for computer use. Flagellar capture will always require that cells are at a dilution where their paths do not frequently cross.

    WIDER IMPLICATIONS OF THE FINDINGS: Combining tracked flagella with mathematical modelling has the potential to reveal new mechanistic insight. By providing the capability as a free-to-use software package, we hope that this ability to accurately quantify the flagellar waveform in large populations of motile cells will enable an abundant array of diagnostic, toxicological and therapeutic possibilities, as well as creating new opportunities for assessing and treating male subfertility.

    STUDY FUNDING/COMPETING INTEREST(S): M.T.G., G.C., J.C.K-B. and D.J.S. gratefully acknowledge funding from the Engineering and Physical Sciences Research Council, Healthcare Technologies Challenge Award (Rapid Sperm Capture EP/N021096/1). J.C.K-B. is funded by a National Institute of Health Research (NIHR) and Health Education England, Senior Clinical Lectureship Grant: The role of the human sperm in healthy live birth (NIHRDH-HCS SCL-2014-05-001). This article presents independent research funded in part by the NIHR and Health Education England. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The data for experimental set (2) were funded through a Wellcome Trust-University of Birmingham Value in People Fellowship Bridging Award (E.H.O.).The authors declare no competing interests.

    Matched MeSH terms: Cell Tracking/methods*
  2. Mok PL, Leow SN, Koh AE, Mohd Nizam HH, Ding SL, Luu C, et al.
    Int J Mol Sci, 2017 Feb 08;18(2).
    PMID: 28208719 DOI: 10.3390/ijms18020345
    Mesenchymal stem cells are widely used in many pre-clinical and clinical settings. Despite advances in molecular technology; the migration and homing activities of these cells in in vivo systems are not well understood. Labelling mesenchymal stem cells with gold nanoparticles has no cytotoxic effect and may offer suitable indications for stem cell tracking. Here, we report a simple protocol to label mesenchymal stem cells using 80 nm gold nanoparticles. Once the cells and particles were incubated together for 24 h, the labelled products were injected into the rat subretinal layer. Micro-computed tomography was then conducted on the 15th and 30th day post-injection to track the movement of these cells, as visualized by an area of hyperdensity from the coronal section images of the rat head. In addition, we confirmed the cellular uptake of the gold nanoparticles by the mesenchymal stem cells using transmission electron microscopy. As opposed to other methods, the current protocol provides a simple, less labour-intensive and more efficient labelling mechanism for real-time cell tracking. Finally, we discuss the potential manipulations of gold nanoparticles in stem cells for cell replacement and cancer therapy in ocular disorders or diseases.
    Matched MeSH terms: Cell Tracking
  3. Ude CC, Shamsul BS, Ng MH, Chen HC, Norhamdan MY, Aminuddin BS, et al.
    Tissue Cell, 2012 Jun;44(3):156-63.
    PMID: 22402173 DOI: 10.1016/j.tice.2012.02.001
    Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 μmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7±0.4 and 14.6±0.5; unlabeled samples had 13.8±0.5 and 15.4±0.6 respectively. Upon staining with 2, 4 and 8 μmol PKH26, BMSCs had retentions of 40.0±5.8, 60.0±2.9 and 95.0±2.9%, while ADSCs had 92.0±1.2, 95.0±1.2 and 98.0±1.2%. ADSCs retentions were significantly higher at 2 and 4 μmol. On dye retention comparison at 8 μmol and 4 μmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0±1.2% to 90.0±0.6% and ADSCs from 94.0±1.2% to 52.0±1.2% (p<0.05) after 24h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p<0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 μmol and BMSCs is 8 μmol, and they can be tracked up to 49 days.
    Matched MeSH terms: Cell Tracking/methods*
  4. Ramli K, Gasim AI, Ahmad AA, Htwe O, Mohamed Haflah NH, Law ZK, et al.
    Tissue Eng Part A, 2019 10;25(19-20):1438-1455.
    PMID: 30848172 DOI: 10.1089/ten.TEA.2018.0279
    We investigated the efficacy of a muscle-stuffed vein (MSV) seeded with neural-transdifferentiated human mesenchymal stem cells as an alternative nerve conduit to repair a 15-mm sciatic nerve defect in athymic rats. Other rats received MSV conduit alone, commercial polyglycolic acid conduit (Neurotube®), reverse autograft, or were left untreated. Motor and sensory functions as well as nerve conductivity were evaluated for 12 weeks, after which the grafts were harvested for histological analyses. All rats in the treatment groups demonstrated a progressive increase in the mean Sciatic Functional Index (motor function) and nerve conduction amplitude (electrophysiological function) and showed positive withdrawal reflex (sensory function) by the 10th week of postimplantation. Autotomy, which is associated with neuropathic pain, was severe in rats treated with conduit without cells; there was mild or no autotomy in the rats of other groups. Histologically, harvested grafts from all except the untreated groups exhibited axonal regeneration with the presence of mature myelinated axons. In conclusion, treatment with MSV conduit is comparable to that of other treatment groups in supporting functional recovery following sciatic nerve injury; and the addition of cells in the conduit alleviates neuropathic pain. Impact Statement It is shown that pretreated muscle-stuffed vein conduit is comparable to that of commercial nerve conduit and autograft in supporting functional recovery following peripheral nerve injury. The addition of neural-differentiated mesenchymal stem cells in the conduit is shown to alleviate neuropathic pain.
    Matched MeSH terms: Cell Tracking
  5. Ude CC, Sulaiman SB, Min-Hwei N, Hui-Cheng C, Ahmad J, Yahaya NM, et al.
    PLoS One, 2014;9(6):e98770.
    PMID: 24911365 DOI: 10.1371/journal.pone.0098770
    In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model.
    Matched MeSH terms: Cell Tracking
  6. Ude CC, Shamsul BS, Ng MH, Chen HC, Ohnmar H, Amaramalar SN, et al.
    Exp Gerontol, 2018 04;104:43-51.
    PMID: 29421350 DOI: 10.1016/j.exger.2018.01.020
    BACKGROUND: Hyaline articular cartilage, which protects the bones of diarthrodial joints from forces associated with load bearing, frictions, and impacts has very limited capacities for self-repair. Over the years, the trend of treatments has shifted to regenerations and researchers have been on the quest for a lasting regeneration. We evaluated the treatment of osteoarthritis by chondrogenically induced ADSCs and BMSCs for a long time functional recovery.

    METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography.

    RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance.

    CONCLUSIONS: Both ADSCs and BMSCs had increased chondrogenic gene expressions using TGF-β3 and BMP-6. The treated knees had improved cartilage scores; PKH26 can provide elongated tracking, while EMG results revealed improved joint recoveries. These could be suitable therapies for osteoarthritis.

    Matched MeSH terms: Cell Tracking
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